Interfering with viral entry is a promising strategy for interrupting the HIV-1 life cycle. Ideally, HIV-1 fusion should be studied under the most physiologically relevant conditions possible, including the use of actual virions and primary cells. We developed a sensitive and specific HIV-1 virion-based assay that permits the study of fusion of virions containing (B-lactamase-Vpr to primary target cells loaded with CCF2, a fluorogenic B-lactamase substrate. We hypothesize that the study of the fusion of HIV-1 virions to biologically relevant target cells, including subsets of primary CD4 T lymphocytes and dendritic cells (DCs) varying in their state of maturation or activation, will reveal important differences that centrally contribute to viral pathogencsis. In Specific Aim 1, we will analyze HIV-1 fusion to subsets of primary CD4 T lymphocytes in human tonsillar tissue. Our preliminary studies show higher levels of HIV-1 fusion to memory than to naive CD4 T cells. Similarly, the state of cellular activation could also influence the susceptibility of cells to fusion. We will investigate whether these differences reflect changes in chemokine coreceptor expression or changes in HIV-1 binding. In Specific Aim 2, we will investigate whether DC maturation and HIV tropism influence the kinetics of HIV-1 virion fusion to DCs and the compartment from which fusion occurs. We have observed dramatic differences in the susceptibility of mature and immature DCs to fusion of R5- and X4-tropic HIV-1 virions. We will characterize the fusion kinetics and investigate the possibility that HIV-1 virions fuse from endosomal compartments. In parallel, we will continue to explore the susceptibility of a wide range of in vivo DCs to X4- and R5-tropic HIV-1 fusion. In Specific Aim 3, we will compare fusion of HIV-1 virions that were transmitted across the mucosal epithelia versus those that were not transmitted. For these studies, we will adapt the virion-based fusion assay to study primary isolates. These virions will be amplified from donor-recipient pairs of HIV-1-infected patients. Adaptation of the assay for analysis of primary isolates is a technological leap forward that will be useful for future studies. The proposed studies promise to advance our understanding of HIV-1 fusion and the cellular determinants that govern this key viral entry event. The findings could suggest new strategies to block this early step in the HIV-1 life cycle and to interrupt the horizontal transmission of HIV-1 in vivo. [unreadable] [unreadable]